Anthriscus Sylvestris
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J Wood Sci (2002) 48:536-541 The Japan Wood Research Society 2002 Shiro Suzuki Norikazu Sakakibara Toshiaki Umezawa Mikio Shimada Survey and enzymatic formation of lignans of Anthriscus sylvestris Received: July 25, 2001 / Accepted: December 22, 2001 Abstract Gas chromatography - mass spectrometry ana- cal properties I-3 and various biological activities (e.g., anti- lysis of the fl-glucosidase-treated MeOH extracts of tumor, antimitotic, antiviral2-S). Moreover, biosynthesis of Anthriscus sylvestris showed, based on comparison of the some lignans is closely related to heartwood formation, mass spectra and retention times with those of authentic which is a metabolic event specific to woody plants but not samples, the presence of lignans, yatein, secoisolaricire- to herbaceous plantsY sinol, lariciresinol, matairesinol, hinokinin, and pluviato- An aryltetralin lignan, podophyllotoxin, isolated from lide. The existence of small amounts of bursehernin was PodophyIlum plants ~-8 is well known to have antitumor ac- suggested by mass chromatography. In addition, nemerosin tivity. The lignan isolated from Podophyllum hexandrurn is and deoxypodophyllotoxin were tentatively identified by exploited commercially as a source of the semisynthetic comparing the mass spectra with those reported in the lit- anticancer drugs etoposide and teniposide. 2-8'1~ However, erature. Enzyme preparations from A. sylvestr& catalyzed owing to the limited supply of the plant, much attention the formation of secoisolariciresinol and lariciresinol from has been focused on the availability and biosynthesis of coniferyl alcohol. Furthermore, the enzyme preparation the lignan, a Several woody plants (e.g., Juniperus savina, catalyzed the formation of lariciresinol from (2)_ Thujopsis dolabrata, Callitris drurnmondii, Hernandia pinoresinols and the formation of secoisolariciresinol from ovigera) have been known to produce podophyllotoxin or (2)-lariciresinols. Thus, pinoresinol/lariciresinol reductase its congeners. 7 Thus, biotechnological production of the (PLR) activity was detected. Chiral high-performance antitumor lignan by the woody plants is a challenging sub- liquid chromatography analysis showed selective formation ject in the field of wood chemistry and biochemistry. As of (+)-lariciresinol and (-)-secoisolariciresinol from (2)_ the first step, it is critically important to understand the pinoresinols with the A. sylvestris PLR preparation, indicat- biosynthetic mechanisms. However, much remains un- ing that the stereochemical property of A. sylvestris known about the biosynthesis of podophyllotoxin. PLR-catalyzed reduction was similar to those of Forsythia In addition to these woody plants and Podophyllum spp., PLR and Arctium lappa ripening seed PLR. some herbaceous plants such as Linum spp. (especially those belonging to the section Syllinum, including Linum Key words Lignan - Anthriscus sylvestris Biosynthesis flavum and Linum album) 5m'12 and Anthriscus sylvestris 13 19 have been known to produce significant amounts of podophyllotoxin congeners. Studies of lignan biosynthesis in Linum spp., 2~ which produce principally 5- Introduction methoxypodophyllotoxin in addition to podophyllotoxin, 2~ have been reported. Although isolation of podophyllotoxin Biosynthesis of lignans has been studied from many aspects from A. sylvestris has not yet been reported, angeloyl because this class of compounds has peculiar stereochemi- podophyllotoxin has been isolated from the species. 22 In addition, this species produces not only the precursor lignans of podophyllotoxin, deoxypodophyllotoxin (= S. Suzuki N. Sakakibara - T. Umezawa (~) - M. Shimada desoxypodophyllotoxin, anthricin) 23 and yatein, 24 but also Wood Research Institute, Kyoto University, Uji, Kyoto 611-0011, the typical heartwood lignans yatein and hinokinin, t7 which Japan Tel. +81-774-38-3628; Fax +81-774-38-3682 are found specifically in the heartwood region in the coni- e-mail: [email protected]~u.ac.jp fers Libocedrus yateensis 25 and Charnaecyparis obtusa, 9 respectively. Thus, knowledge of the lignan biosynthetic Part of this report was presented at the 43rd Lignin Symposium,Fuchu, mechanism in A. sylvestris can be applied to biotechnolo- October 1998 gical production of podophyllotoxin in woody and herba- 537 OH H3CO.~OH H3C O~.,~-.r.-- ~ min by a linear solvent gradient protocol of CH3CN-H20 at .o 2o. t = 0 (23:77) to 15 min, then to 50:50 at t = 20min, and held at this ratio for an additional 5 min. (2) Solvent system B was used for gradient elution at i ml/min by a linear solvent o o. ~" OCH3 OH gradient protocol of CH3CN-H20 at t = 0-6 min from 15:85 Lariciresinol Secoiso- Matairesinol to 17:83; at t = 6-16rain from t7:83 to 20:80; 20:80 at t = L\ I~ lariciresinol 16-20min; at t = 20-26 from 20:80 to 50 : 50; and held at this "~ "OCH3 ratio for an additional 5min. Chiral HPLC separation of OH lariciresinol and secoisolariciresinol was conducted exactly O~ as previously described) ~ Silica gel thin-layer chromatogra- 2Jo L,;o phy (TLC) and silica gel column chromatography employed CH30 Kieselgel 60 F254 (Merck) and Kieselgel 60 (Merck), respec- ~0 tively. All solvents and reagents used were of reagent or . .3 "g5~ 0 HPLC grade unless otherwise mentioned. OR2 H3CO~ q RI=OCH3, R2=CH3,Yatein Hinokinin Nemerosin 0 RI=H, R2=H, Pluviatolide Compounds RI=H, R2=CH3, Bursehernin [9,9-2H2, OC2H3]Coniferyl alcohol,27 (+)-[9,9,9',9'- OH 2H4]pinoresinols,2S (-+)-pinoresinols, 27 (+)-[9,9,9',9'- 2H4]lariciresinols,28 (2_-)-lariciresinols, > (_+)-[9,9,9',9'-aH4] secoisolariciresinols,28 (--)-secoisolariciresinols,27 (-)- matairesinols, 3~ (+)-hinokinins, 9 (+)-pluviatolides, 9 and ( + )-bursehernins 28 were prepared previously. ( _+)-Yateins Oeoxypodo- OCH3 were prepared from (+)-4-demethylyatein benzyl ethers (a phyllotoxin Podophyllotoxin gift of Dr. Shingo Kawai) by debenzylation (10% Pd/C, H2) followed by methylation (diazomethane). Fig. 1. Chemical structures of lignans (_+)-Yateins, ~H-NMR (CDC13): 2.43-2.63 (3H, m, C7:8,s,H), 2.88 (1H, dd, J = 13.8 and 6.3, C7H), 2.92 (1H, dd, J = 13.8 and 5.4, CTH), 3.817 (3H, s, OCH3), 3.819 (6H, s, ceous plants and to studies of heartwood lignan biosynthetic OCH 3 • 2), 3.87 (1H, dd, J = 9.0 and 7.6, C9H), 4.17 (1H, mechanisms. In addition, this species exhibits good growth dd, J = 9.1 and 7.2, CgH), 5.92-5.93 (2H, m, -OCH20-), 6.35 behavior and can be maintained easily in laboratories. (2H, s, C2,6H), 6.45-6.47 (2H, m, C2,,6,H), 6.69 (1U, dd, J = Altogether, A. sylvestris is probably a good plant mate- 0.8 and 7.6, Cs,H). rial for lignan biosynthesis studies to access mechanisms involved in the formation of antitumor and heartwood lignans. As the first step, it is necessary to characterize the Plant materials precursor lignans of yatein and deoxypodophyllotoxin in A. sylvestris. We report here the preliminary survey of lignans Anthriscus sylvestris (L.) Hoffm. (Umbelliferae) plants in this species and the enzyme activities catalyzing forma- were grown from seeds collected in a suburb of Brussels, tion of lariciresinol and secoisolariciresinol, which are up- Belgium in 1996 or at Kyoto University Forest in Ashiu, stream lignans in the biosynthetic pathway (Fig. 1). Kyoto in 1997. The plants were maintained in the experi- mental garden of the Wood Research Institute, Kyoto Uni- versity for about 6 months. Materials and methods Lignans in Anthriscus sylvestris Instrumentation Aerial parts (including leaves, petioles, racemes) and roots ~H-nuclear magnetic resonance (NMR) spectra were ob- of A. sylvestris were individually harvested and washed with tained with a JNM-LA400MK FT NMR system (JEOL) tap and distilled water. Each sample (0.7-1.6 g) was frozen using tetramethylsilane as an internal standard. Chemical (liquid N2), freeze-dried, disintegrated with scissors, and shifts and coupling constants (J) are given in d and Hertz, then extracted with hot MeOH (11 ml). An aliquot (5 mg) of respectively. Gas chromatography-mass spectrometry (GC- the MeOH extract was treated with fi-glucosidase (from MS) was conducted as previously described. 26 Reversed- almonds; Sigma G-0395) (10 units in 0.5 ml of a 0.1 M NaOAc phase high-performance liquid chromatography (HPLC) buffer at pH 5.0) for 24h at 37~ The reaction mixture was was conducted as previously described 27 with the following extracted with CH2C12 (0.5 ml • 2), and the combined solu- elution conditions: The column was a Waters Novapak C1s tion was evaporated off. The resulting residue was subjected (150 • 3.9mm), which was eluted with two solvent systems: to GC-MS analysis after derivatization with 7,al of N,O- (1) Solvent system A was used for gradient elution at i ml/ bis(trimethylsilyl) acetamide (BSA) for 45 rain at 60~ 538 In a separate experiment, aerial parts of A. sylvestris of the BSA solution was subjected to GC-MS analysis, and (from University Forest in Ashiu) were harvested and the lignans formed were identified and quantified (Table 1). freeze-dried as above. The dried materials (48.7g) were ground with a Waring blender and extracted with hot Enzymatic conversion of (• MeOH (100ml • 10); then the solvent was evaporated off. and (• The MeOH extract (12.3 g) was dispersed in Et20 (50ml • 10), and the Et20-soluble materials were removed. The The reaction mixture consisted of 150/~l of 50mM of Et20-insoluble residue (10.3 g) was suspended in 180ml of (+)-[9,9,9',9'-2H4]pinoresinols in MeOH, lml of 50mM 0.1M NaOAc buffer (pH 5.0) containing 2544 units of/3- NADPH in a 0.1M potassium phosphate buffer (pH 7.0), glucosidase, and the solution was incubated for 24h at 37~ and 16mi of the ceil-free extracts from A. sylvestris leaves. Then the solution was extracted with EtOAc (80ml • 4), The reaction was initiated by adding (+)-[9,9,9',9'-2H4] and the solvent was evaporated off. The EtOAc extract pinoresinols. After incubating for 1 h at 30~ the reaction (721mg) was fractionated into six fractions by silica gel mixture was extracted with EtOAc containing unlabeled column (q~ 30 • 80ram) chromatography.